Method for processing lipoaspirate cells

ABSTRACT

Cells present in processed lipoaspirate tissue are used to treat patients. Methods of treating patients include processing adipose tissue to deliver a concentrated amount of stem cells obtained from the adipose tissue to a patient. The methods may be practiced in a closed system so that the stem cells are not exposed to an external environment prior to being administered to a patient. Compositions that are administered to a patient include a mixture of adipose tissue and stem cells so that the composition has a higher concentration of stem cells than when the adipose tissue was removed from the patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application No.Ser. 10/316,127, Entitled SYSTEMS AND METHODS FOR TREATING PATIENTS WITHPROCESSED LIPOASPIRATE CELLS, and filed Dec. 9, 2002 now abandoned,which claims the benefit of U.S. Provisional Application No. Ser.60/338,856, entitled BEDSIDE DEVICE, SYSTEM AND USE OF PROCESSEDLIPOASPIRATE CELLS AND ADIPODERIVED STEM CELLS, and filed Dec. 7,2001,the entire contents of which are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention generally relates to cells derived from adipose tissue,and more particularly, to adipo-derived stem cells, methods of usingadipo-derived stem cells, compositions containing adipo-derived stemcells, and systems for preparing and using adipo-derived stem cells.

2. Description of Related Art

Regenerative medicine can be defined as harnessing the body'sregenerative mechanisms in a clinically targeted manner, using them inways that are not part of the normal healing mechanism or byartificially amplifying normal mechanisms. One classic example of thisprocess is found in bone marrow transplantation where hematopoietic stemand progenitor cells are harvested from a donor and placed into arecipient in whom the normal hematopoietic regenerative mechanisms havebeen ablated or substantially depleted or impaired, thereby replacing orregenerating the blood-forming capacity of the recipient (Thomas 1994).In recent clinical and pre-clinical studies this approach has beenextended to the non-hematopoietic stem cell component of bone marrowwith studies regenerating (or attempting to regenerate) tissuesincluding bone (Connolly 1998; Horwitz, Prockop et al. 1999; Horwitz,Prockop et al. 2001), heart (Fukuda 2001; Orlic, Kajstura et al. 2001;Orlic, Kajstura et al. 2001; Strauer, Brehm et al. 2002), and liver(Avital, Inderbitzin et al. 2001). These studies have been based on thedetection of the presence of non-hematopoietic stem cells andendothelial precursor cells in bone marrow (Prockop, Azizi et al. 2000)(Pittenger, Mackay et al. 1999) (Shi, Rafii et al. 1998; Carmeliet andLuttun 2001).

These studies used bone marrow transplant recipient animals in whichdonor and host cells could be distinguished by genetic markers to showthat some fraction of new blood vessel development in the recipients wasderived from the donor marrow cells (Carmeliet and Luttun 2001)(Takahashi, Kalka et al. 1999; Murayama, Tepper et al. 2002). While thiswork definitively demonstrates that marrow contains such cells it hasgenerally been extended to mean that marrow is therefore the only tissuethat contains relevant numbers of such cells to the extent that when aninvestigator detects endothelial precursor cells (EPCs) or marrow stemcells (MSCs) in the circulation it is automatically assumed that thesecells are necessarily marrow-derived. Thus, the concept that cellpopulations from other tissues might represent an alternative or perhapssuperior source of therapeutically relevant cell populations is notaddressed.

It has been demonstrated that adipose tissue contains a populationmultipotent stem cells (Huang, Beanes et al. 2002; Mizuno, Zuk et al.2002) (Zuk, Zhu et al. 2001). Zuk et al. (Zuk et al., (In Press) HumanAdipose Tissue Is A Source Of Multipotent Stem Cells, Molecular Biologyof the Cell) and others have previously shown that this tissue is asource of endothelial cells (Kern, Knedler et al. 1983; Hutley,Herington et al. 2001) [U.S. Pat. No. 5,372,945 Alchas et al, 1994]though these latter documents did not examine and do not speak in anyway to endothelial precursor cells.

Stem cells are the master cells of the body. Stem cells from embryos orembryonic stem cells (ESCs) are know to become many if not all of thecell and tissue types of the body. These early fetal cells not onlycontain all the genetic information of the individual but also containthe nascent capacity to become any of the 200+ cells and tissues of thebody. Ongoing research suggests that these cells have tremendousscientific and clinical potential.

However, ESCs have theoretic limitations to their use. If usedclinically they would necessarily be derived from another individual, anembryo. When stem cells or tissues derived from them are transplantedinto another person, toxic immune suppressing drugs may be needed by thecell recipient to prevent rejection. In addition, another individual'scells can carry viruses or other rare but significant diseases that canbe transmitted to the recipient. Also, ESC-like cells (eg. teratomas)are known to form tumors.

Recently, non-embryonic or adult stem cells have been identified andrepresent an important potential alternative to the clinical use ofESCs. These cells reside quietly in many if not all tissues, presumablywaiting to respond to trauma or other destructive disease processes sothat they can heal the injured tissue. Emerging scientific evidenceindicates that each individual carries a pool of stem cells that mayshare with ESCs the ability to become many if not all types of cells andtissues.

Adult stem cell populations have been shown to be present in one or moreof skin, muscle, marrow, liver, brain, and adipose tissue. To dateproposed application of such cells in tissue engineering involveincreasing cell number, purity, and maturity by processes of cellpurification and cell culture. These steps are necessary to compensatefor the rarity of stem cells in most tissues. For example, mesenchymalstem cell frequency in bone marrow is estimated at between 1 in 100,000and 1 in 1 million nucleated cells. Similarly, extraction of stem cellsfrom skin involves a complicated series of cell culture steps overseveral weeks. Use of skeletal muscle-derived stem cells in clinicaltrials of heart disease employs a two to three week culture phase inwhich cell number is increased to clinically relevant numbers and celldifferentiation into muscle is promoted.

These expansion and differentiation steps may provide increased cellnumber, purity, and maturity, but they do so at a cost. This cost caninclude one or more of: loss of cell function due to cell aging, loss ofpotentially useful non-stem cell cell populations, delays in potentialapplication of cells to patients, increased monetary cost, and increasedrisk of contamination of cells with environmental microorganisms duringculture. While human data is now becoming available with marrow-derivedcells that have not been manipulated but rather used as essentiallywhole marrow (Horwitz, Prockop et al. 1999; Horwitz, Prockop et al.2001) (Strauer, Brehm et al. 2002), the clinical benefit derived hasbeen suboptimal, an outcome almost certainly related to the limited celldose and purity available from marrow.

A number of devices have been developed for harvesting cells fromadipose tissue, but these devices can suffer from one or more ofinability to optimally accommodate an asperation device for removal ofadipose tisssue, lack of partial or full automation from the harvestingof adipose tissue phase through the processing of tissue phases, lack ofvolume capacity greater than 100 ml of adipose tissue, lack of apartially or completely closed system from the harvesting of adiposetissue phase through the processing of tissue phases, and lack ofdisposabilty of components to attenuate concomitant risks ofcross-contamination of material from one sample to another.

There is need for alternate approaches in which a population of activecells with increased yield, consistency and/or purity can be preparedrapidly and reliably, and whereby the need for post-extractionmanipulation of the cells can be reduced or eliminated. Ideally thiscell population would be obtained in a manner that is suitable for theirdirect placement into a recipient.

SUMMARY OF THE INVENTION

The present invention is directed to compositions, methods, and systemsfor using cells derived from adipose tissue that are placed directlyinto a recipient along with such additives necessary to promote,engender, or support a therapeutic, structural, or cosmetic benefit.

In one embodiment, adipose tissue processing occurs in a system thatmaintains a closed, sterile fluid/tissue pathway. This is achieved byuse of a pre-assembled, linked set of closed, sterile containers andtubing allowing for transfer of tissue and fluid elements within aclosed pathway. This processing set can be linked to a series ofprocessing reagents (e.g., saline, enzymes, etc.) inserted into a devicewhich can control the addition of reagents, temperature, and timing ofprocessing thus relieving operators of the need to manually manage theprocess. In a preferred embodiment the entire procedure from tissueextraction through processing and placement into the recipient would allbe performed in the same facility, indeed, even within the same room ofthe patient undergoing the procedure.

In accordance with one aspect of the invention, raw adipose tissue isprocessed to substantially remove mature adipocytes and connectivetissue thereby obtaining a heterogeneous plurality of adiposetissue-derived cells suitable for placement within the body of arecipient. The cells may be placed into the recipient in combinationwith other cells, tissue, tissue fragments, or other stimulators of cellgrowth and/or differentiation. In a preferred embodiment, the cells,with any of the above mentioned additives, are placed into the personfrom whom they were obtained in the context of a single operativeprocedure with the intention of deriving a therapeutic, structural, orcosmetic benefit to the recipient.

In one embodiment, a method of treating a patient includes steps of: a)providing a tissue removal system; b) removing adipose tissue from apatient using the tissue removal system, the adipose tissue having aconcentration of stem cells; c) processing at least a pact of theadipose tissue to obtain a concentration of stem cells other than theconcentration of stem cells of the adipose tissue before processing; andd) administering the stem cells to a patient without removing the stemcells from the tissue removal system before being administered to thepatient.

In another embodiment, a method of treating a patient includes: a)providing an adipose tissue removal system; b) removing adipose tissuefrom a patient using the adipose tissue removal system, the adiposetissue having a concentration of stem cells; c) processing the adiposetissue to increase the concentration of stem cells in the adiposetissue; d) mixing the adipose tissue having the concentrated stem cellswith another unitportion of adipose tissue; and e) administering theadipose tissue with the increased concentration of stem cells to apatient.

A system in accordance with the invention herein disclosed includes a) atissue collection container including i) a tissue collecting inlet portstructured to receive adipose tissue removed from a patient; and ii) afilter disposed within the container and being structured to retainadipose tissue removed from a patient and to pass non-adipose tissueremoved from the patient; b) a mixing container coupled to the tissuecollection container to receive stem cells obtained from the adiposetissue without removal of the stem cells from the tissue removal system,and including an additive port for the administration of at least oneadditive to mix with the stem cells contained therein; and c) an outletstructured to permit the cells in the mixing container to be removedfrom the tissue collection system for administration to a patient.

A composition of the invention includes a first portion of adiposetissue removed from a patient that has a concentration of stem cells,and a second portion of adipose tissue removed from the patient having aconcentration of stem cells greater than the first portion of adiposetissue.

Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. Additional advantages and aspects ofthe present invention are apparent in the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a tissue removal system for processing adipose tissue.

FIG. 2 depicts a tissue collection container of the tissue removalsystem of FIG. 1.

FIG. 3 is a partial cross-sectional view of the tissue collectioncontainer of FIG. 2.

FIG. 4 depicts a processing device for automating the operation of atissue removal system.

FIG. 5 is a graph depicting graft weight versus cell dose.

FIG. 6 is a graph depicting the effects of processed lipoaspirate onalcohol treated mice

FIG. 7 is a photomicrograph of liver tissue of an alcohol treated mousethat received processed lipoaspirate.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Reference will now be made in detail to the presently preferredembodiments of the invention, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same or similar referencenumbers are used in the drawings and the description to refer to thesame or like parts. It should be noted that the drawings are insimplified form and are not to precise scale. In reference to thedisclosure herein, for purposes of convenience and clarity only,directional terms, such as, top, bottom, left, right, up, down, over,above, below, beneath, rear, and front, are used with respect to theaccompanying drawings. Such directional terms should not be construed tolimit the scope of the invention in any manner.

Although the disclosure herein refers to certain illustratedembodiments, it is to be understood that these embodiments are presentedby way of example and not by way of limitation. The intent of thefollowing detailed description, although discussing exemplaryembodiments, is to be construed to cover all modifications,alternatives, and equivalents of the embodiments as may fall within thespirit and scope of the invention as defined by the appended claims. Thepresent invention may be practiced in conjunction with various cell ortissue seperation techniques that are conventionally used in the art,and only so much of the commonly practiced process steps are includedherein as are necessary to provide an understanding of the presentinvention.

The present invention is directed to a cell population present inadipose tissue, and systems and methods for administering the cellpopulation into a human or animal patient. The cell population of theadipose tissue may be used as a source of cells for therapeutic andcosmetic applications. Among other things, the cells may be used forregenerative medicine, such as diseases that can be treated withregenerating cells. The cells of the population may be administered to apatient without other adipocytes or connective tissue, or may beadministered mixed together with adipose tissue in a concentratedamount, as discussed herein.

It has been discovered that adipose tissue is an especially rich sourceof stem cells. This finding may be due, at least in part, to the ease ofremoval of the major non-stem cell component of adipose tissue, theadipocyte. Thus, in both human and animal studies, processedlipoaspirate (PLA) contains stem cells at a frequency of at least 0.1%,and more typically greater than 0.5%. In certain embodiments of theinvention, PLA has been obtained which contains between about 2-12% stemcells. In even further embodiments, the PLA is processed to obtain apopulation of cells where the stem cells constitute between up to 100%of the cells in the population. The amount of stem cells obtained inaccordance with the invention herein disclosed is substantially greaterthan the published frequency of 1 in 100,000 (0.001%) in marrow(Castro-Malaspina, Ebell et al. 1984) (Muschler, Nitto et al. 2001).Furthermore, collection of adipose tissue is associated with lowermorbidity than collection of a similar volume of marrow (Nishimori,Yamada et al. 2002). In addition, adipose tissue contains endothelialprecursor cells, which are capable of providing therapy to patients (seee.g., Masuda, H., C. Kalka, and T. Asahara, Endothelial progenitor cellsfor regeneration. Hum Cell, 2000. 13(4): p. 153-60; Kaushal, S., et al.,Functional small-diameter neovessels created using endothelialprogenitor cells expanded ex vivo. Nat Med, 2001. 7(9): p. 1035-40; andKawamoto, A., et al., Therapeutic potential of ex vivo expandedendothelial progenitor cells for myocardial ischemia. Circulation, 2001.103(5): p. 634-7.

As used herein, “adipose tissue” refers to a tissue containing multiplecell types including adipocytes and microvascular cells. Adipose tissueincludes stem cells and endothelial precursor cells. Accordingly,adipose tissue refers to fat including the connective tissue that storesthe fat.

As used herein, “unit of adipose tissue” refers to a discrete ormeasurable amount of adipose tissue. A unit of adipose tissue may bemeasured by determining the weight and/or volume of the unit. Based onthe data identified above, a unit of processed lipoaspirate, as removedfrom a patient, has a cellular component in which at least 0.1% of thecellular component is stem cells. In reference to the disclosure herein,a unit of adipose tissue may refer to the entire amount of adiposetissue removed from a patient, or an amount that is less than the entireamount of adipose tissue removed from a patient. Thus, a unit of adiposetissue may be combined with another unit of adipose tissue to form aunit of adipose tissue that has a weight or volume that is the sum ofthe individual units.

As used herein, “portion” refers to an amount of a material that is lessthan a whole. A minor portion refers to an amount that is less than 50%,and a major portion refers to an amount greater than 50%. Thus, a unitof adipose tissue that is less than the entire amount of adipose tissueremoved from a patient is a portion of the removed adipose tissue.

As used herein, “stem cell” refers to a multipotent cell with thepotential to differentiate into a variety of other cell types, whichperform one or more specific functions and have the ability toself-renew. Some of the stem cells disclosed herein may be pluripotent.

As used herein, “processed lipoaspirate” (PLA) refers to adipose tissuethat has been processed to separate the active cellular component (e.g.,the component containing stem cells) from the mature adipocytes andconnective tissue. Typically, PLA refers to the pellet of cells obtainedby washing and separating the cells from the adipose tissue. The pelletis typically obtained by centrifuging a suspension of cells so that thecells aggregate at the bottom of a centrifuge container.

In practicing the methods disclosed herein, the cells that areadministered to a patient are obtained from adipose tissue. Adiposetissue can be obtained by any method known to a person of ordinary skillin the art. For example, adipose tissue may be removed from a patient bysuction-assisted lipoplasty, ultrasound-assisted lipoplasty, andexcisional lipectomy. In addition, the procedures may include acombination of such procedures, such as a combination of excisionallipectomy and suction-assisted lipoplasty. As the tissue or somefraction thereof is intended for reimplantation into a patient theadipose tissue should be collected in a manner that preserves theviability of the cellular component and that minimizes the likelihood ofcontamination of the tissue with potentially infectious organisms, suchas bacteria and/or viruses. Thus, the tissue extraction should beperformed in a sterile or aseptic manner to minimize contamination.Suction assisted lipoplasty may be desirable to remove the adiposetissue from a patient as it provides a minimally invasive method ofcollecting tissue with minimal potential for stem cell damage that maybe associated with other techniques, such as ultrasound assistedlipoplasty.

For suction-assisted lipoplastic procedures, adipose tissue is collectedby insertion of a cannula into or near an adipose tissue depot presentin the patient followed by aspiration of the adipose into a suctiondevice. In one embodiment, a small cannula may be coupled to a syringe,and the adipose tissue may be aspirated using manual force. Using asyringe or other similar device may be desirable to harvest relativelymoderate amounts of adipose tissue (e.g., from 0.1 ml to several hundredmilliliters of adipose tissue). Procedures employing these relativelysmall devices have the advantage that the procedures can be performedwith only local anesthesia, as opposed to general anesthesia. Largervolumes of adipose tissue above this range (e.g., greater than severalhundred millilters) may require general anesthesia at the discretion ofthe donor and the person performing the collection procedure. Whenlarger volumes of adipose tissue are desired to be removed, relativelylarger cannulas and automated suction devices may be employed in theprocedure.

Excisional lipectomy procedures include, and are not limited to,procedures in which adipose tissue-containing tissues (e.g., skin) isremoved as an incidental part of the procedure; that is, where theprimary purpose of the surgery is the removal of tissue (e.g., skin inbariatric or cosmetic surgery) and in which adipose tissue is removedalong with the tissue of primary interest.

The adipose tissue that is removed from a patient is collected into adevice for further processing. As discussed herein, and in oneembodiment, the device is designed for and dedicated to the purpose ofcollecting tissue for manufacture of a processed adipose tissue cellpopulation, which includes stem cells and/or endothelial precursorcells. In other embodiments, the device may be any conventional devicethat is typically used for tissue collection by physicians performingthe extraction procedure.

The amount of tissue collected will be dependent on a number ofvariables including, but not limited to, the body mass index of thedonor, the availability of accessible adipose tissue harvest sites,concomitant and pre-existing medications and conditions (such asanticoagulant therapy), and the clinical purpose for which the tissue isbeing collected. Experience with transplant of hematopoietic stem cells(bone marrow or umbilical cord blood-derived stem cells used toregenerate the recipient's blood cell-forming capacity) shows thatengraftment is cell dose-dependent with threshold effects. Thus, it islikely that the general principle that “more is better” will be appliedwithin the limits set by other variables and that where feasible theharvest will collect as much tissue as possible.

It has been discovered that the stem cell percentage of 100 ml ofadipose tissue extracted from a lean individual is greater than thatextracted from an obese donor (Table 1). This reflects a dilutive effectof the increased fat content in the obese individual. Therefore, it maybe desirable, in accordance with one aspect of the invention, to obtainlarger amounts of tissue from overweight donors compared to the amountsthat would be withdrawn from leaner patients. This observation alsoindicates that the utility of this invention is not limited toindividuals with large amounts of adipose tissue.

TABLE 1 Effect of Body Mass Index on Tissue and Cell Yield Body MassAmount of Tissue Total Cell Yield Index Status Obtained (g) (×10⁷)Normal 641 ± 142 2.1 ± 0.4 Obese 1,225 ± 173   2.4 ± 0.5 p value 0.030.6

Patients undergoing treatment in accordance with the disclosure hereinreceive a different concentration of stem cells than other treatmentsemploying adipose tissue or stem cells derived from adipose tissue.Thus, the adipose tissue that is removed from a patient is processed tochange the concentration of stem cells that are administered to thepatient. In a preferred embodiment of the invention, patients receive ahigher concentration of stem cells than the concentration of stem cellstypically present in adipose tissue transplants and other similar stemcell based therapies. The concentrated stem cells may be administered ina composition comprising adipo-derived stem cells and/or endothelialprecursor cells substantially free from mature adipocytes and connectivetissue, or, as another example, the concentrated stem cells may beadministered in a composition comprising a unit of adipose tissue withan increased amount of stem cells. A composition of the inventionincludes a concentration of stem cells that is greater than theconcentration of stem cells found in an equivalent unit of non-processedadipose tissue. In certain embodiments, the composition has a cellularcomponent in which at least 0.1% of the cells are stem cells. In otherembodiments, the composition has a cellular component in which the stemcells comprise between about 2% and 12% of the cellular component.Higher concentrations of stem cells, such as up to 100%, are alsoincluded in different compositions. The composition may includeadditional components, such as cell differentiation factors, growthpromoters, immunosuppressive agents, or medical devices, as discussedherein. To obtain certain compositions in which the compositionprimarily contains one type of cell (e.g., adipo-derived stem cells oradipo-derived endothelial precursor cells), any suitable method forseparating the different cell types may be employed, such as the use ofcell-specific antibodies that recognize and bind antigens present oneither stem cells or endothelial precursor cells.

For most applications preparation of the active cell population willrequire depletion of the mature fat-laden adipocyte component of adiposetissue. This is typically achieved by a series of washing anddisaggregation steps in which the tissue is first rinsed to reduce thepresence of free lipids (released from ruptured adipocytes) andperipheral blood elements (released from blood vessels severed duringtissue harvest), and then disaggregated to free intact adipocytes andother cell populations from the connective tissue matrix. In certainembodiments, the entire adipocyte component, or non-stem cell component,is separated from the stem cell component of the adipose tissue. Inother embodiments, only a portion or portions of the adipocyte componentis separated from the stem cells. Thus, in certain embodiments, the stemcells can be administered with endothelial precursor cells.

Rinsing is an optional, but preferred, step in which the tissue is mixedwith solutions to wash off free lipid and single cell components, suchas those components in blood, leaving behind intact adipose tissuefragments. In one embodiment, the adipose tissue that is removed fromthe patient is mixed with isotonic saline or other physiologicsolution(s) (e.g., Plasmalyte®, of Baxter Inc or Normoso® of AbbottLabs). Intact adipose tissue fragments can be separated from the freelipid and cells by any means known to persons or ordinary skill in theart including, but not limited to, filtration, decantation,sedimentation, or centrifugation. In the illustrated embodiment of theinvention, the adipose tissue is separated from non-adipose tissue byemploying a filter disposed within a tissue collection container, asdiscussed herein. In other embodiments, the adipose tissue is separatedfrom non-adipose tissue using a tissue collection container thatutilizes decantation, sedimentation, and/or centrifugation techniques toseparate the materials.

The intact tissue fragments are then disaggregated using anyconventional techniques or methods, including mechanical force (mincingor shear forces), enzymatic digestion with single or combinatorialprotelolytic enzymes, such as collagenase, trypsin, lipase, liberase H1,as disclosed in U.S. Pat. No. 5,952,215, and pepsin, or a combination ofmechanical and enzymatic methods. For example, the cellular component ofthe intact tissue fragments may be disaggregated by methods usingcollagenase-mediated dissociation of adipose tissue, similar to themethods for collecting microvascular endothelial cells in adiposetissue, as disclosed in U.S. Pat. No. 5,372,945. Additional methodsusing collagenase that may be used in practicing the invention aredisclosed in U.S. Pat. Nos. 5,830,714 and 5,952,215, and by Williams, S.K., S. McKenney, et al. (1995). “Collagenase lot selection andpurification for adipose tissue digestion.” Cell Transplant 4(3): 281-9.Similarly, a neutral protease may be used instead of collagenase, asdisclosed in Twentyman, P. R. and J. M. Yuhas (1980). “Use of bacterialneutral protease for disaggregation of mouse tumours and multicellulartumor spheroids.” Cancer Lett 9(3): 225-8. Furthermore, methods mayemploy a combination of enzymes, such as a combination of collagenaseand trypsin, as disclosed in Russell, S. W., W. F. Doe, et al. (1976).“Inflammatory cells in solid murine neoplasms. I. Tumor disaggregationand identification of constituent inflammatory cells.” Int J Cancer18(3): 322-30; or a combination of an enzyme, such as trypsin, andmechanical dissociation, as disclosed in Engelholm, S. A., M.Spang-Thomsen, et al. (1985). “Disaggregation of human solid tumours bycombined mechanical and enzymatic methods.” Br J Cancer 51(1): 93-8.

The active cell population (processed lipoaspirate) may then be obtainedfrom the disaggregated tissue fragments by reducing the presence ofmature adipocytes. A suspension of the processed lipoaspirate and theliquid in which the adipose tissue was disaggregated is then passed toanother container, such as a cell collection container. The suspensionmay flow through one or more conduits to the cell collection containerby using a pump, such as a peristaltic pump, that withdraws thesuspension from the tissue collection container and urges it to the cellcollection container. Other embodiments may employ the use of gravity ora vacuum while maintaining a closed system. Separation of the cells inthe suspension may be achieved by buoyant density sedimentation,centrifugation, elutriation, differential adherence to and elution fromsolid phase moieties, antibody-mediated selection, differences inelectrical charge; immunomagnetic beads, flourescence activated cellsorting (FACS), or other means. Examples of these various techniques anddevices for performing the techniques may be found in Hemstreet, G. P.,3rd, P. G. Enoch, et al. (1980). “Tissue disaggregation of human renalcell carcinoma with further isopyknic and isokinetic gradientpurification.” Cancer Res 40(4): 1043-9; Schweitzer, C. M., van, et al.(1995). “Isolation and culture of human bone marrow endothelial cells.”Exp Hematol 23(1): 41-8; Gryn, J., R. K. Shadduck, et al. (2002).“Factors affecting purification of CD34(+) peripheral blood stem cellsusing the Baxter Isolex 300i.” J Hematother Stem Cell Res 11(4): 719-30;Prince, H. M., J. Bashford, et al. (2002). “Isolex 300i CD34-selectedcells to support multiple cycles of high-dose therapy.” Cytotherapy4(2): 137-45; Watts, M. J., T. C. Somervaille, et al. (2002). “Variableproduct purity and functional capacity after CD34 selection: a directcomparison of the CliniMACS (v2.1) and Isolex 300i (v2.5) clinical scaledevices.” Br J Haematol 118(1): 117-23; Mainwaring, G. and A. F. Rowley(1985). “Separation of leucocytes in the dogfish (Scyliorhinus canicula)using density gradient centrifugation and differential adhesion to glasscoverslips.” Cell Tissue Res 241(2): 283-90; Greenberg, A. W. and D. A.Hammer (2001). “Cell separation mediated by differential rollingadhesion.” Biotechnol Bioeng 73(2): 111-24; and U.S. Pat. Nos.6,277,060; 6,221,315; 6,043,066; 6,451,207; 5,641,622; and 6,251,295. Inthe illustrated embodiment, the cells in the suspension are separatedfrom the acellular component of the suspension using a spinning membranefilter. In other embodiments, the cells in the suspension are separatedfrom the acellular component using a centrifuge. In one such exemplaryembodiment, the cell collection container may be a flexible bag that isstructured to be placed in a centrifuge (e.g., manually or by robotics).In other embodiments, a flexible bag is not used. After centrifugation,the cellular component forms a pellet, which may then be resuspendedwith a buffered solution so that the cells can be passed through one ormore conduits to a mixing container, as discussed herein. Theresuspension fluids may be provided by any suitable means. For example,a buffer may be injected into a port on the cell collection container,or the cell collection container may include a reserve of buffer thatcan be mixed with the pellet of cells by rupturing the reserve. When aspinning membrane filter is used, resuspension is optional since thecells remain in a volume of liquid after the separation procedure.

Although certain embodiments of the invention are directed to methods offully disaggregating the adipose tissue to separate the active cellsfrom the mature adipocytes and connective tissue, additional embodimentsof the invention are directed to methods in which the adipose tissue isonly partially disaggregated. For example, partial disaggregation may beperformed with one or more enzymes, which are removed from the at leasta part of the adipose tissue early, relative to an amount of time thatthe enzyme would otherwise be left thereon to fully disaggregate thetissue. Such a process may require less processing time.

In one particular embodiment, the tissue is washed with sterile bufferedisotonic saline and incubated with collagenase at a collagenaseconcentration, temperature, and time sufficient to provide adequatedisaggregation. In a preferred embodiment, the collagenase enzyme usedwill be approved for human use by the relevant authority (e.g., the U.S.Food and Drug Administration). Suitable collagenase preparations includerecombinant and non-recombinant collagenase. Non-recombinant collagenasemay be obtained from F. Hoffmann-La Roche Ltd, Indianapolis, Ind. and/orAdvance Biofactures Corp., Lynbrook, N.Y. Recombinant collagenase mayalso be obtained as disclosed in U.S. Pat. No. 6,475,764.

In one embodiment, solutions contain collagenase at concentrations fromabout 10 μg/ml to about 50 μg/ml and are incubated at from about 30° C.to about 38° C. for from about 20 minutes to about 60 minutes. Theseparameters will vary according to the source of the collagenase enzyme,optimized by empirical studies, in order to validate that the system iseffective at extracting the desired cell populations in an appropriatetime frame. A particular preferred concentration, time and temperatureis 20 μg/ml collagenase (Blendzyme 1, Roche) incubated for 45 minutes,at about 37° C. In a particularly preferred embodiment the collagenaseenzyme used is material approved for human use by the relevant authority(e.g., the U.S. Food and Drug Administration). The collagenase usedshould be free of micro-organisms and contaminants, such as endotoxin.

Following disaggregation the active cell population may be washed/rinsedto remove additives and/or by-products of the disaggregation process(e.g., collagenase and newly-released free lipid). The active cellpopulation could then be concentrated by centrifugation or other methodsknown to persons of ordinary skill in the art, as discussed above. Thesepost-processing wash/concentration steps may be applied separately orsimultaneously.

In one embodiment, the cells are concentrated and the collagenaseremoved by passing the cell population through a continuous flowspinning membrane system or the like, such as, for example, the systemdisclosed in U.S. Pat. Nos. 5,034,135; and 5,234,608.

In addition to the foregoing, there are many post-wash methods that maybe applied for further purifying the active cell population. Theseinclude both positive selection (selecting the target cells), negativeselection (selective removal of unwanted cells), or combinationsthereof.

In one embodiment, a solid phase material with adhesive propertiesselected to allow for differential adherence and/or elution of asubpopulation of cells within the processed lipoaspirate is insertedinto the system after the cell washing step. This general approach hasbeen performed in clinical blood transfusion in which filtersdifferentially capturing leukocytes are used to deplete transfused redcells of contaminating white blood cell (Soli, M., et al., A multicentreevaluation of a new filtration protocol for leucocyte depletion ofhigh-haematocrit red blood cells collected by an automated bloodcollection system. Vox Sang, 2001. 81(2): p. 108-12; Smith, J. W.,Apheresis techniques and cellular immunomodulation. Ther Apher, 1997.1(3): p. 203-6). Filters of this type are distributed by Pall Bedical(Leukogard RS and Purecell RCQ) and Asahi (RS2000). Differentialadherence has also been applied to positive selection of monocytes(Berdel, W. E., et al., Purification of human monocytes by adherence topolymeric fluorocarbon. Characterization of the monocyte-enriched cellfraction. Immunobiology, 1982. 163(5): p. 511-20) and epidermal stemcells (Bickenbach, J. R. and E. Chism, Selection and extended growth ofmurine epidermal stem cells in culture. Exp Cell Res, 1998. 244(1): p.184-95). In this embodiment the processed lipoaspirate would be passedthrough a filter material under flow and buffer conditionspre-determined to promote differential adherence of target cells andunwanted cell populations. For positive selection the filter materialand conditions would allow preferential adherence of target cells whileunwanted material would pass freely through the filter and be washedaway with excess buffer. Target cells would be eluted from the filter bychanging the conditions such as flow rate, pH, ionic strength, and/orpresence of cations necessary for adhesion. The filter material could bein the form of a three-dimensional mesh, packed cassette of smallparticles, hollow-fibers or other mechanism with high surface area. In apreferred embodiment, this filter device would be an integral part ofthe disposable set shown in FIG. 1 and would be inserted into the deviceshown in FIG. 4. Both the set and device would have to be modifiedslightly from those examples shown in the specified figures; FIG. 1 toinclude the filter and housing and FIG. 4 to allow for insertion of thefilter housing and tubing (including valves) necessary for maintenanceof a closed, sterile fluid pathway. Alternatively the mixing chamber(Component 108 of FIG. 4; component 30 of FIG. 1) could be replaced bythe device fittings and filter/housing respectively.

An alternate embodiment of this differential adherence approach wouldinclude use of antibodies and/or combinations of antibodies recognizingsurface molecules differentially expressed on target and unwanted cells.Selection on the basis of expression of specific cell surface markers(or combinations thereof) is another commonly applied technique in whichantibodies are attached (directly or indirectly) to a solid phasesupport structure (Geiselhart, A., et al., Positive selection of CD56+lymphocytes by magnetic cell sorting. Nat Immun, 1996. 15(5): p. 227-33;Formanek, M., et al., Magnetic cell separation for purification of humanoral keratinocytes: an effective method for functional studies withoutprior cell subcultivation. Eur Arch Otorhinolaryngol, 1998. 255(4): p.211-5; Graepler, F., U. Lauer, and M. Gregor, Magnetic cell sorting forparietal cell purification using a new monoclonal antibody withoutinfluence on cell function. J Biochem Biophys Methods, 1998. 36(2-3): p.143-55; Kobari, L., et al., CD133+ cell selection is an alternative toCD34+ cell selection for ex vivo expansion of hematopoietic stem cells.J Hematother Stem Cell Res, 2001. 10(2): p. 273-81; Mohr, M., et al.,Simultaneous immunomagnetic CD34+ cell selection and B-cell depletion inperipheral blood progenitor cell samples of patients suffering fromB-cell non-Hodgkin's lymphoma. Clin Cancer Res, 2001. 7(1): p. 51-7; andPugh, R. E., et al., CD19 selection improves the sensitivity of B celllymphoma detection. J Hematother, 1998. 7(2): p. 159-68). This approachhas obvious applications in both positive and negative selection inwhich, for example, residual white blood cells might be removed by useof the CD45 antibody). Similarly, Reyes et al have applied a complexblend of antibodies in the selection of a multipotential adultprogenitor cell from human bone marrow (Reyes, M., et al., Purificationand ex vivo expansion of postnatal human marrow mesodermal progenitorcells. Blood, 2001. 98(9): p. 2615-25). For example, an antibody such asAP2 (Joyner, C. J., et al., Development of a monoclonal antibody to theaP2 protein to identify adipocyte precursors in tumours of adiposedifferentiation. Pathol Res Pract, 1999. 195(7): p. 461-6) whichspecifically binds to adipocytic cells could be employed topreferentially deplete residual adipocytic cells (including immatureadipocytes and adipoblasts). Positive selection could be applied by useof antibodies specific for the target cell population(s). For example,Quirici et al have used antibodies to the Nerve Growth Factor Receptorto enrich bone marrow-derived mesenchymal stem cells (Quirici, N., etal., Isolation of bone marrow mesenchymal stem cells by anti-nervegrowth factor receptor antibodies. Exp Hematol, 2002. 30(7): p. 783-91).

In one embodiment of an antibody-based approach, an antibody (forexample AP2) or a cocktail of antibodies (for example AP2, CD3, CD19,CD11b) would be added to the processed lipoaspirate. Many otherantibodies and combinations of antibodies will be recognized by oneskilled in the art and these examples are provided by way of exampleonly. After incubation, under conditions pre-determined to allow foroptimal binding of these antibodies to their cognate antigens, the cellswould be washed by passing through the spinning membrane filter or otherembodiment of the cell washing chamber to remove unbound, excessantibody. The cells would then be passed over a solid phase structuresimilar to that described in the embodiment above but in which the solidphase has attached a secondary antibody capable of high affinityattachment to the primary antibodies now bound to the cell surface.Target cells, for example the adipose tissue-derived stem cell, wouldpass freely through this filter by virtue of the absence of expressionof cell surface antigens recognized by the selected antibody (antibodycocktail) thereby creating a negative selection system. In thisembodiment the disposable set (FIG. 3) and device (FIG. 4) would besubject to minor modifications very similar to those described in theabove embodiment.

An antibody-mediated positive selection embodiment could be achieved invery similar fashion by including a third additive that facilitatesdetachment of the cells from the solid phase support. In thisembodiment, the enzyme papain or cymopapain could be added to cleave theantibody molecules and release cells from the solid phase support(Civin, C. I., et al., Positive stem cell selection—basic science. ProgClin Biol Res, 1990. 333(387): p. 387-401; discussion 402). Anotheralternative would be the use of specific peptides that would competewith the cell surface antigen for binding to the antibodies, asdescribed by Tseng-Law et al, U.S. Pat. No. 6,017,719.

In another embodiment the cell pellet could be resuspended, layered over(or under) a fluid material formed into a continuous or discontinuousdensity gradient and placed in a centrifuge for separation of cellpopulations on the basis of cell density. Examples of media suitable forformation of such gradients include Percoll and Ficoll-Paque (Qian, X.,L. Jin, and R. V. Lloyd, Percoll Density Gradient-Enriched Populationsof Rat Pituitary Cells: Interleukin 6 Secretion, Proliferative Activity,and Nitric Oxide Synthase Expression. Endocr Pathol, 1998. 9(1): p.339-346; Smits, G., W. Holzgreve, and S. Hahn, An examination ofdifferent Percoll density gradients and magnetic activated cell sorting(MACS) for the enrichment of fetal erythroblasts from maternal blood.Arch Gynecol Obstet, 2000. 263(4): p. 160-3) or Ficoll-Paque (Lehner, M.and W. Holter, Endotoxin-free purification of monocytes for dendriticcell generation via discontinuous density gradient centrifugation basedon diluted Ficoll-Paque Plus. Int Arch Allergy Immunol, 2002. 128(1): p.73-6). Van Merris et al, (Van Merris, V., et al., Separation of bovinebone marrow into maturation-related myeloid cell fractions. Vet ImmunolImmunopathol, 2001. 83(1-2): p. 11-7) employed a discontinuousthree-step Percoll gradient to separate bovine myeloid cells accordingto their maturation state on this basis. This embodiment would becapable of separating out certain residual blood cell populations andimmature adipocytes (pre-adipocytes) from the cell population.

In a similar embodiment continuous flow approaches such as apheresis(Smith, J. W., Apheresis techniques and cellular immunomodulation. TherApher, 1997. 1(3): p. 203-6) and elutriation (with or withoutcounter-current) (Lasch, J., G. Kullertz, and J. R. Opalka, Separationof erythrocytes into age-related fractions by density or size?Counterflow centrifugation. Clin Chem Lab Med, 2000. 38(7): p. 629-32;Ito, Y. and K. Shinomiya, A new continuous-flow cell separation methodbased on cell density: principle, apparatus, and preliminary applicationto separation of human buffy coat. J Clin Apheresis, 2001. 16(4): p.186-91; Dlubek, D., et al., Enrichment of normal progenitors incounter-flow centrifugal elutriation (CCE) fractions of fresh chronicmyeloid leukemia leukapheresis products. Eur J Haematol, 2002. 68(5): p.281-8) may also be employed. Such mechanisms have been used tofractionate blood cells, including separation of red blood cells on thebasis of age (Lasch, J., G. Kullertz, and J. R. Opalka, Separation oferythrocytes into age-related fractions by density or size? Counterflowcentrifugation. Clin Chem Lab Med, 2000. 38(7): p. 629-32) andapplication of this general approach to further purification of cells ofinterest from processed lipoaspirate will be readily apparent to oneskilled in the art. This embodiment may require modification of thedevice in FIG. 4 and the disposable set (FIG. 3) such that the devicewould be integrated with a second device providing the apheresis orelutriation capability.

Adherence to plastic followed by a short period of cell expansion hasalso been applied in bone marrow-derived adult stem cell populations(Jaiswal, N., et al., Osteogenic differentiation of purified,culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem,1997. 64(2): p. 295-312; Hou, L., et al., Study of in vitro expansionand differentiation into neuron-like cells of human umbilical cord bloodmesenchymal stem cells. Zhonghua Xue Ye Xue Za Zhi, 2002. 23(8): p.415-9). This approach uses culture conditions to preferentially expandone population while other populations are either maintained (andthereby reduced by dilution with the growing selected cells) or lost dueto absence of required growth conditions. Sekiya et al have describedconditions which might be employed in this regard for bonemarrow-derived stem cells (Sekiya, I., et al., Expansion of Human AdultStem Cells from Bone Marrow Stroma: Conditions that Maximize the Yieldsof Early Progenitors and Evaluate Their Quality. Stem Cells, 2002.20(6): p. 530-41). This approach (with or without differential adherenceto the tissue culture plastic) could be applied to a further embodimentof this invention. In this embodiment the cells are removed from thedevice shown in FIG. 4 and placed into a second device providing thecell culture component. This could be in the form of a conventionallaboratory tissue culture incubator or a Bioreactor-style device such asthat described by Tsao et al, U.S. Pat. No. 6,001,642, or by Armstronget al, U.S. Pat. No. 6,238,908. In an alternative embodiment, the mixingcomponent (component 108 of the device shown in FIG. 4; component 30 inFIG. 3) could be replaced by a Bioreactor component allowing forshort-term adherence and/or cell culture of the processed lipoaspirate.This alternate embodiment would permit integration of the Bioreactorcomponent to the device and remove the need for removing the cells fromthis device and placement within another.

In certain embodiments, the active cell population is administereddirectly into the patient. In other words, the active cell population(e.g., the stem cells and/or endothelial precursor cells) areadministered to the patient without being removed from the system orexposed to the external environment of the system before beingadministered to the patient. Providing a closed system reduces thepossibility of contamination of the material being administered to thepatient. Thus, processing the adipose tissue in a closed system providesadvantages over existing methods because the active cell population ismore likely to be sterile. In such an embodiment, the only time the stemcells and/or endothelial precursor cells are exposed to the externalenvironment, or removed from the system, is when the cells are beingwithdrawn into an application device and being administered to thepatient. In one embodiment, the application device can also be part ofthe closed system. Thus, the cells used in these embodiments are notprocessed for culturing, or cryopreserved.

The active cells that have been concentrated, as described above, may beadministered to a patient without further processing, or may beadministered to a patient after being mixed with other tissues or cells.In certain embodiments, the concentrated active cells (e.g., stem cellsor endothelial precursor cells) are mixed with one or more units ofadipose tissue that has not been similarly processed. Thus, bypracticing the methods of the invention, a composition comprisingadipose tissue with an enhanced concentration of active cells may beadministed to the patient. The volumes of the various units of adiposetissue may be different. For example, one volume may be at least 25%greater than the volume of another unit of adipose tissue. Furthermore,one volume may be at least 50%, such as at least 100%, and even 150% ormore greater than the volume of another unit of adipose tissue. Inaddition, the desired composition may be obtained by mixing a first unitof adipose tissue with the concentrated active cell population, whichmay be a cell pellet containing the active cells, with one or more otherunits of adipose tissue. In certain embodiments, these other units willnot have an increased concentration of stem cells, or in other words,will have an active cell concentration less than that contained in thefirst unit of adipose tissue. In other embodiments, one of the units iscryopreserved material that contains, for example, an increasedconcentration of active cells.

In other embodiments, at least a portion of the active cell populationis stored for later implantation/infusion. The population may be dividedinto more than one aliquot or unit such that part of the population ofstem cells and/or endothelial precursor cells is retained for laterapplication while part is applied immediately to the patient. Moderateto long-term storage of all or part of the cells in a cell bank is alsowithin the scope of this invention, as disclosed in U.S. patentapplication Ser. No. 10/242,094, entitled PRESERVATION OF NON EMBRYONICCELLS FROM NON HEMATOPOIETIC TISSUES, filed Sep. 12, 2002, which claimsthe benefit of U.S. Provisional Patent Application 60/322,070 filed Sep.14, 2001, which is commonly assigned, and the contents of which areexpressly incorporated herein by reference. In such an embodiment, thecells may be mixed with one or more units of fresh or preserved adiposetissue to provide a composition containing the stem cells at a higherconcentration than a unit of adipose tissue prior to processing.

At the end of processing, the concentrated cells may be loaded into adelivery device, such as a syringe, for placement into the recipient byeither subcutaneous, intravenous, intramuscular, or intraperitonealtechniques. In other words, cells may be placed into the patient by anymeans known to persons of ordinary skill in the art, for example, theymay be injected into blood vessels for systemic or local delivery, intotissue (e.g., cardiac muscle, or skeletal muscle), into the dermis(subcutaneous), into tissue space (e.g., pericardium or peritoneum), orinto tissues (e.g., periurethral emplacement), or other location.Preferred embodiments include placement by needle or catheter, or bydirect surgical implantation in association with additives such as apreformed matrix.

The active cell population may be applied alone or in combination withother cells, tissue, tissue fragments, demineralized bone, growthfactors such as insulin or drugs such as members of the thiaglitazonefamily, biologically active or inert compounds, resorbable plasticscaffolds, or other additive intended to enhance the delivery, efficacy,tolerability, or function of the population. The cell population mayalso be modified by insertion of DNA or by placement in cell culture insuch a way as to change, enhance, or supplement the function of thecells for derivation of a cosmetic, structural, or therapeutic purpose.For example, gene transfer techniques for stem cells are known bypersons of ordinary skill in the art, as disclosed in Mosca, J. D., J.K. Hendricks, et al. (2000). “Mesenchymal stem cells as vehicles forgene delivery.” Clin Orthop(379 Suppl): S71-90, and may include viraltransfection techniques, and more specifically, adeno-associated virusgene transfer techniques, as disclosed in Walther, W. and U. Stein(2000). “Viral vectors for gene transfer: a review of their use in thetreatment of human diseases.” Drugs 60(2): 249-71, and Athanasopoulos,T., S. Fabb, et al. (2000). “Gene therapy vectors based onadeno-associated virus: characteristics and applications to acquired andinherited diseases (review).” Int J Mol Med 6(4): 363-75. Non-viralbased techniques may also be performed as disclosed in Muramatsu, T., A.Nakamura, et al. (1998). “In vivo electroporation: a powerful andconvenient means of nonviral gene transfer to tissues of living animals(Review).” Int J Mol Med 1(1): 55-62.

In one aspect, the cells could be mixed with unprocessed fragments ofadipose tissue and placed back into the recipient using a very largegauge needle or liposuction cannula. Transfer of autologous fat withoutsupplementation with processed cells is a common procedure in plasticand reconstructive surgery. However, results can be unpredictable as thetransferred material tends to rapidly reabsorb resulting in an unstablegraft. Adipose tissue-derived cells of the invention that are, forexample, substantially depleted of mature adipocytes may provide anenvironment that supports prolonged survival and function of the graft.

In another aspect, the cell population could be placed into therecipient and surrounded by a resorbable plastic sheath such as thatmanufactured by MacroPore Biosurgery, Inc. (U.S. Pat. Nos. 6,269,716 and5,919,234). In this setting the sheath would prevent prolapse of muscleand other soft tissue into the area of a bone fracture thereby allowingthe emplaced processed adipose tissue-derived cells to promote repair ofthe fracture. In this aspect, the beneficial effect might be enhanced bysupplementation with additional components such as pro-osteogenicprotein growth factors or biological or artificial scaffolds.

In another aspect, the cells could be combined with a gene encoding apro-osteogenic growth factor which would allow cells to act as their ownsource of growth factor during bone healing or fusion. Addition of thegene could be by any technology known in the art including but notlimited to adenoviral transduction, “gene guns,” liposome-mediatedtransduction, and retrovirus or lentevirus-mediated transduction.

Particularly when the cells and/or tissue containing the cells areadministered to a patient other than the patient from which the cellsand/or tissue were obtained, one or more immunosuppressive agents may beadministered to the patient receiving the cells and/or tissue to reduce,and preferably prevent, rejection of the transplant. Examples ofimmunosuppressive agents suitable with the methods disclosed hereininclude agents that inhibit T-cell/B-cell costimulation pathways, suchas agents that interfere with the coupling of T-cells and B-cells viathe CTLA4 and B7 pathways, as disclosed in U.S. Patent Pub. No.20020182211. Other examples include cyclosporin, myophenylate mofetil,rapamicin, and anti-thymocyte globulin.

In certain embodiments of the invention, the cells are administered to apatient with one or more cellular differentiation agents, such ascytokines and growth factors. Examples of various cell differentiationagents are disclosed in Gimble, J. M., C. Morgan, et al. (1995). “Bonemorphogenetic proteins inhibit adipocyte differentiation by bone marrowstromal cells.” J Cell Biochem 58(3): 393-402; Lennon, D. P., S. E.Haynesworth, et al. (1995). “A chemically defined medium supports invitro proliferation and maintains the osteochondral potential of ratmarrow-derived mesenchymal stem cells.” Exp Cell Res 219(1): 211-22;Majumdar, M. K., M. A. Thiede, et al. (1998). “Phenotypic and functionalcomparison of cultures of marrow-derived mesenchymal stem cells (MSCs)and stromal cells.” J Cell Physiol 176(1): 57-66; Caplan, A. I. and V.M. Goldberg (1999). “Principles of tissue engineered regeneration ofskeletal tissues.” Clin Orthop(367 Suppl): S12-6; Ohgushi, H. and A. I.Caplan (1999). “Stem cell technology and bioceramics: from cell to geneengineering.” J Biomed Mater Res 48(6): 913-27; Pittenger, M. F., A. M.Mackay, et al. (1999). “Multilineage potential of adult humanmesenchymal stem cells.” Science 284(5411): 143-7; Caplan, A. I. and S.P. Bruder (2001). “Mesenchymal stem cells: building blocks for molecularmedicine in the 21st century.” Trends Mol Med 7(6): 259-64; Fukuda, K.(2001). “Development of regenerative cardiomyocytes from mesenchymalstem cells for cardiovascular tissue engineering.” Artif Organs 25(3):187-93; Worster, A. A., B. D. Brower-Toland, et al. (2001).“Chondrocytic differentiation of mesenchymal stem cells sequentiallyexposed to transforming growth factor-beta1 in monolayer andinsulin-like growth factor-I in a three-dimensional matrix.” J OrthopRes 19(4): 738-49; Zuk, P. A., M. Zhu, et al. (2001). “Multilineagecells from human adipose tissue: implications for cell-based therapies.”Tissue Eng 7(2): 211-28; and Mizuno, H., P. A. Zuk, et al. (2002).“Myogenic differentiation by human processed lipoaspirate cells.” PlastReconstr Surg 109(1): 199-209; discussion 210-1.

By administering the stem cells and/or endothelial precursor cells to apatient, one can treat numerous diseases, including, and not limited to,bone-related disorders, diseases, or injuries, including slow/non-unionfractures, osteoporosis (age-related or chemotherapy-induced), inheriteddiseases of bone (osteogenesis imperfecta); adipose related disorders ordiseases; liver related diseases, disorders, or injuries, includingliver failure, hepatitis B, and hepatitis C; myocardial infarctions,including heart attack or chronic heart failures; renal diseases orkidney damage; retinal diseases or damage or necrosis; wound healing(e.g., from surgery or diabetic ulcers); skeletal muscle disorders bothtraumatic and inherited; cartilege and joint repair both traumatic andautoimmune; lung injuries; diabetes; intestinal disorders; nervoussystem disorders, dieseases, or injuries, such as central nervoussystems disorders, diseases, or injuries, including spinal cordinjuries, Parkinson's disease, Alzheimer's disease, and stroke.

The stem cells may also be administered to a patient for cosmeticpurposes, such as by enhancing or improving physical features, includingreducing wrinkles, enhancing organ mass, and the like.

A tissue removal system for removing adipose tissue from a patient isillustrated in FIG. 1. In a broad embodiment, tissue removal system 10includes a tissue collecting container 12 and a mixing container 30coupled to the tissue collecting container 12. The coupling betweenmixing container 30 and tissue collecting container 12 preferablydefines a closed system in which the tissue that is directed from tissuecollecting container 12 to mixing container 30 is not exposed to theexternal environment. System 10 also includes an outlet 32 that isstructured to permit concentrated stem cells to be removed from tissuecollection system 10 to be administered to a patient. The tissuecollection container 12 includes a tissue collecting inlet port 14 and afilter 16. Filter 16 is disposed within the container, and is structuredto retain adipose tissue and to pass non-adipose tissue as, for example,the tissues are removed from the patient. More specifically, filter 16allows passage of free lipid, blood, and saline, while retainingfragments of adipose tissue during, or in another embodiment after, theinitial harvesting of the adipose tissue. In that regard, filter 16includes a plurality of pores, of either the same or different sizes,but ranging in size from about 20 μm to 5 mm. In a preferred embodiment,the filter is a medical grade polyester mesh of around 200 μm thicknesswith a pore size of around 265 μm and around 47% open area. Thismaterial holds the tissue during rinsing but allows cells to pass outthrough the mesh following tissue disaggregation. Thus, when the tissuesare aspirated from the patient, the non-adipose tissue may be separatedfrom the adipose tissue. Mixing container 30 includes an additive port31 that is structured to allow a user to administer an additive to themixing container 30 to mix with stem cells contained in the mixingcontainer 30. In a preferred embodiment, the dimensions of the tissuecollection container 12 should be such as to allow retention ofapproximately 1 liter of tissue fragments within the filter. In otherembodiments, the tissue collection container 12 may be sized to hold agreater or smaller volume of tissue fragments; for example, the tissuecollection container may be sized to store at least 100 mL of adiposetissue fragments, and up to about 2 L of adipose tissue fragments.

Referring to additional features present in system 10 of FIG. 1, tissueinlet port 14 is coupled to cannula 24 by way of tubing 22 to define atissue removal line. In the illustrated embodiment, cannula 24 is anintegrated, single-use liposuction cannula, and the tubing is a flexibletubing. The cannula is dimensioned to be inserted into a patient toremove adipose tissue from the patient. The tubing 22 used in the systemshould be capable of withstanding negative pressure associated withsuction assisted lipoplasty to reduce the likelihood of collapsing.Tissue collection container 12 also includes an aspiration port 18disposed on the opposite side of filter 16 from tissue inlet port 14.Aspiration port 18 is structured to be coupled to a suction device 20,which may be manually or automatically operated. Suction device 20 maybe a syringe or may be an electric vacuum, among other things. Suctiondevice 20 should be capable of providing a sufficient negative pressureto container 12 and cannula 24 to aspirate tissue from a patient. Asillustrated, suction device 20 is coupled to aspiration port 18 by wayof tubing 22.

Tissue removal system 10 is illustrated as also including a cellcollection container 26 positioned between tissue collection container12 and mixing container 30. Cell collection container 26 is positionedwithin system 10 so that cells, such as stem cells, pass from tissuecollection container 12 to the cell collection container 26 before beingpassed to mixing container 30. In the illustrated embodiment, cellcollection container 26 is coupled to tissue collection container 12 byway of cell collecting port 48. In one embodiment of system 10, cellcollection container 26 includes a cell concentrator (not shown) thatfacilitates separation of the cells in a suspension. An example of acell concentrator is a centrifuge device that may separate cells fromother materials based on, for example, the size or density of the cells.Another example is a spinning membrane filter, as discussed above.System 10 is also illustrated as including a filter 28 structured topass the cells from cell collection container 26 to mixing container 30,and to prevent passage of material that is, for example, larger than,the cells. Cell collection container 26 also includes an outlet to wastecontainer 36. The direction of flow of the material contained in cellcollection container 26 is determined by the positioning of one or morevalves which can control whether the material flows to waste container36 or mixing container 30.

In the illustrated embodiment, cell filter 28 comprises a plurality ofpores having a diameter, or length less than 200 μm. In certainembodiments, the pores may have diameters that are smaller than 200 μm.In other embodiments, the pores have diameters between 20 and 200 μm.Cell filter 28 may be spaced apart from cell collection container 26 ormay be contained within cell collection container 26. Cell filter 28 mayalso be integrally formed in cell collection container 26. Additionalembodiments of system 10 do not include filter 28. Cell collectioncontainer may be fabricated from any suitable material. For example,cell collection container 26 may be a plastic bag, such as thoseconventionally used in processing blood in blood banks; or in otherembodiments, it may be structurally rigid. In certain embodiments, cellcollection container 26 may include a component preparation chamber anda cell washing/separation chamber.

In certain embodiments, the component preparation chamber includes oneor more ports for addition of agents that can enhance the process ofseparating stem cells for administering to a patient, such as growthfactors or buffers for resuspending the cells, as discussed above. Inthese embodiments, component preparation chamber preferably includes amixing device to mix or agitate the cells and additives in thecontainer. Component preparation chamber also includes one or more portsfor removing the cells collected therein. One port may be provided topass the cells toward mixing container 30. Other ports may be providedto direct cells, or a portion of the cells, to other targets, such asimplant materials, including bone fragments, or to cell culturing orpurification devices. In one embodiment, the cell washing/separationchamber includes a spinning membrane filter component, which may be usedas the cell concentrator in addition to or, preferably, as as analternative to a centrifuge device.

System 10 is also illustrated as including a tissue retrieval line 34which is positioned to provide a conduit from tissue collectioncontainer 12 to mixing container 30. Thus, tissue retrieval line 34passes or directs tissue contained within tissue collection container 12to mixing container 30 where the tissue can be mixed with cells obtainedfrom cell collection container 26. In the illustrated embodiment, tissueretrieval line 34 extends into tissue container 12 to remove adiposetissue that is contained in filter 16. Tissue is passed or directedthrough tissue retrieval line 34 using one or more pumps or suctiondevices to pass adipose tissue that has been rinsed, but not necessarilydisaggregated.

In one embodiment, system 10 includes a temperature control device thatis positioned with respect to system 10 to adjust the temperature of thematerial contained in the tissue collection container 12. In certainembodiments, the temperature control device is a heater, and in otherembodiments, temperature control device is a cooler. In additionalembodiments, the temperature control device may be able to switchbetween a heater and a cooler. The temperature control device may be adevice that adjusts the temperature of the adipose tissue contained intissue collecting container 12, or may be a device that is positioned tochange the temperature of fluid being delivered to tissue collectingcontainer 12. It has been found that heating the adipose tissuefacilitates disaggregation of the tissue to enhance the separation ofthe active cell component. In addition, it is desirabile in certainembodiments to cool a portion of the tissue, preferably the active cellcomponent to provide protection to the cells. Even mild cooling of thecells may provide suitable protection to enhance cell survival duringthe processing.

Outlet 32 of tissue removal system 10 is illustrated as being acomponent of mixing container 30. In additional embodiments, outlet 32is spaced apart from mixing container 30. Outlet 32 preferably comprisesa closure that maintains the sealed configuration of tissue removalsystem 10, and in certain embodiments, outlet 32 comprises a fluidimpermeable membrane (e.g., a membrane that is impermeable to liquid andair). Outlet 32 should be structured to pass the composition in mixingcontainer 30 to a patient under the appropriate conditions. For example,if a syringe is used to withdraw the composition, outlet 32 should beable to accommodate a needle of the syringe without compromising thesterility of the system or composition. In additional embodiments, ifthe outlet is coupled to a device that is configured to administer thecomposition, but not to withdraw the composition, such as a cannula thatadministers the composition by applying positive pressure to displacethe composition through the cannula, outlet 32 should be configured toallow the composition contained in mixing container 30 to be passed intothe cannula. In other embodiments, outlet 32 may comprise, or be coupledin a closed-system fashion to, the device for adminstering thecomposition, such as a needle of a syringe or a cannula foradministering the composition by applying positive pressure.

Tissue removal system 10 is also illustrated as including a wastecontainer 36 positioned to collect waste from tissue collectioncontainer 12. In the illustrated embodiment, waste container 36 is alsocoupled and positioned to receive waste from cell collection container26. A wash container 38 is provided in fluid communication with washline 39 to deliver a washing fluid, such as saline or any other suitablebuffer, via wash port 46 to tissue collection container 12. Tissuecollection container 12 also includes an air inlet 40 for controllingthe amount of pressure within tissue collection container 12. Anadditive line 42 is provided on tissue collection container 12 to permitan additive to be added to tissue collection container 12. In referenceto the methods disclosed herein, additive line 42 is provided to deliverone or more enzymes to tissue collection container 12 to facilitate theseparation of the active cell component from the rest of the adiposetissue contained in filter 16. As illustrated, additive line 42comprises a needle 44 which can be used to receive the enzyme from asuitable container.

A particular embodiment of components of tissue removal system 10 areillustrated in FIGS. 2 and 3 where like numbers represent like parts. Inthe particular embodiment of FIGS. 2 and 3, tissue collection container12 includes a body that retains its form when suction is applied to thecontainer. More specifically, tissue collection container 12 includes arigid body, for example, a body constructed of a medical gradepolycarbonate containing a roughly conical filter pocket of medicalgrade polyester with a mesh size of 275 μm. The rigid tissue collectioncontainer may have a size of approximately eight inches high andapproximately five inches in diameter; the wall thickness may be about0.125 inches. The interior of the cylinder is accessed through two portsfor suction tubing, two ports with tubing for connection through steriledocking technology, and two ports for needle puncture access through arubber septum. The same functionality could be achieved with differentmaterials, mesh size, and the number and type of ports. For example,mesh pore sizes smaller than 100 μm or as large as several thousandmicrons would achieve the same purpose of allowing passage of saline andblood cells while retaining adipose tissue aggregates and fragments.Similarly, the device purpose could be achieved by use of an alternativerigid plastic material, by substitution of the disposable cannula with anon-disposable, multi-use sterile cannula, or by many othermodifications that would be known to those skilled in the art However,in other embodiments of tissue removal system 10, tissue collectioncontainer 12 may include a collapsible body, such as a tissue collectionbag. In such systems, the bag is preferably provided with a support,such as an internal or external frame, that helps reduce the likelihoodthat the bag will collapse upon the application of suction to the bag.

In order to reduce contamination within tissue removal system 10, one ormore clamps 23 may be provided on the various lines or conduits tocontrol the flow of material through the lines to the various componentsof the system. Clamps 23 permit a user to effectively seal variousregions of tissue removal system 10. In a preferred embodiment, one ormore of the components of system 10 are disposable. Avoiding reusing thecomponents in this embodiment helps to reduce contamination that may beassociated with repeated use of various components. In addition,providing the components in a disposable set provides an advantage ofbeing able to sterilize all of the components at a single time, whichmay substantially reduce the time required for practicing the methodsdisclosed herein. In fully or partially automoated embodiments,computer-controlled valves may be implemented in addition to or as analternative to clamps 23.

In addition, tissue removal system 10 may include additional devices orcomponents that permit, among other things, determination of the volumeof material retained in the filter 16, to allow recording of writteninformation regarding the extraction or processing procedure, or performother supplementary functions such as attaching the device to a stand orbedding during operation.

The components of the tissue removal system 10 should be made ofmaterials that are non-reactive with biological fluids or tissues, andnon-reactive with agents used in processing biological fluids andtissues. In additon, the materials from which the various components aremade should be capable of withstanding sterilization, such as byautoclaving, and irradiation, including but not limited to beta- orgamma-irradiation. The tubing and the cannula handle may be made of anysuitable material, such as polyethylene. The cannula may be made ofstainless steel.

In accordance with the invention herein disclosed, the tissue removalsystem 10 provides a closed system that is convenient for removal,processing, and administration of stem cells found in adipose tissue.The system can be placed near the patient for removal of adipose tissue,and the tissue can be processed without requiring the tissue to beremoved from the system. Thus, a system is provided can provide freshstem cell enhanced compositions to a patient, and reduces potentialrisks associated with culturing and or preserving stem cells.

Referring to the disclosure herein, a method for extracting tissue froma patient may include the following steps: (i) preparing the patient asfor traditional lipoplasty; (ii) removing the cannula and the tissueremoval system from the packaging materials to the sterile field; (iii)connecting a liposuction pump (with conventional trap and in-linemicrobial filters) to the hose adaptor leading from the tissuecollection container; (iv) ensuring that the tubing screw clamps are notengaged on the suction ports of the tissue collection container; (v)using the cannula as a regular liposuction cannula to remove unwantedadipose tissue; (vi) applying in a manual operation embodiment twotubing screw clamps to seal the tissue collection container after thedesired amount of adipose tissue have been collected with the tissuecollection container; and (vii) ensuring that the tissue collectioncontainer is properly labeled with a patient identification label, andrecording other information on the label (date and time of procedure,etc.) in accordance with institutional practice.

Referring to the illustrated tissue removal system 10, tissue iscollected directly into the processing components by attaching thetubing 22 to the suction source 20 with an in-line fluid trap andinserting the cannula 24 into the harvest site. Adipose tissue is thenaspirated into the tissue collecting container 12 where it is retainedby the filter 16 held within the tissue collection container 12.Following tissue collection the collected adipose tissue can be rinsedwith a washing fluid, such as sterile isotonic saline, contained in washcontainer 38 added to tissue collection container 12 via wash line 39.When the tissue collecting container 12 is made of a rigid material inthe illustrated embodiment to support collection under suction, the airdisplaced from the housing during addition of saline can be ventedthrough the air-inlet port 40. Alternatively the air may be displacedinto the waste container 36 or similar holding place. Once the tissue isrinsed the waste material can be allowed to flow into the wastecontainer 36.

In certain embodiments, units of intact adipose tissue may be removedfrom tissue collection container 12 prior to disaggregating the adiposetissue in collection container 12. The units of intact adipose tissuemay be passed along tissue retrieval line 34 so that the units can bedelivered to mixing container 30. In these embodiments, the intacttissue can be mixed with the stem cells prior to administration to apatient.

After the tissue has been collected, needle 44 can be inserted into asterile vial of collagenase-containing enzyme solution which is thenpassed into tissue collection container 12 where it is mixed with theadipose tissue at or around 37° C. for 30-60 minutes. Washing steps maybe repeated as needed and the disaggregated tissue may be washedfollowing elution of the active cell population in order to maximizeyield. At the end of tissue disaggregation the tissue collectioncontainer 12 is placed upright to allow flotation of the adipocytes. Theactive cell population is then allowed to flow into cell collectioncontainer 26 where the cells are separated from collagenase and residualfree lipid. Cells may be washed and/or concentrated by any method knownto persons of ordinary skill in the art including but not limited tosequential centrifugation/re-suspension washes or continuous flowmechanisms. The concentrated, washed cells are then allowed to flow intomixing container 30 where they can be mixed with intact tissue fromtissue retrieval line 34 and/or any intended additives before beingremoved through the outlet 32 for administration to a patient. Thematerial contained in cell collecting container 26 may be filtered usingcell filter 28 following washing to enhance removal of unwanted residualcell and tissue aggregates that could lead to embolism upon application.

During the processing, one or more additives may be added to the variouscontainers as needed to enhance the results. Some examples of additivesinclude agents that optimize washing and disaggregation, additives thatenhance the viability of the active cell population during processing,anti-microbial agents (e.g., antibiotics), additives that lyseadipocytes and/or red blood cells, or additives that enrich for cellpopulations of interest (by differential adherence to solid phasemoieties or to otherwise promote the substantial reduction or enrichmentof cell populations).

In the above embodiment, the tissue collecting container 12 is intrinsicto the processing components of the tissue removal system 10.Alternatively a separate tissue collecting container, such as thatdescribed in patent application Ser. No. 10/242,094, entitledPRESERVATION OF NON EMBRYONIC CELLS FROM NON HEMATOPOIETIC TISSUES,filed Sep. 12, 2002, which claims the benefit of U.S. Provisional PatentApplication 60/322,070 filed Sep. 14, 2001, which is commonly assigned,and the contents of which are expressly incorporated herein by referencecould be employed in whole or in part with subsequent transference ofthe disaggregated material to the processing components. Additionalpotential tissue collecting containers are disclosed in U.S. Pat. Nos.6,316,247 and 5,372,945.

As indicated above, in certain embodiments of the invention, the methodsmay be automated by providing one or more additional devices that canautomatically perform the steps of the methods. In such embodiments, aprocessing device (e.g., microprocessor or personal computer) is adevice to partially or completely automate the steps described above.Examples of steps amenable to such automation include, but are notlimited to, controlling the ingress and egress of fluids and tissuesalong particular tubing paths by controlling pumps and valves of thesystem or processing device; detecting blockages with pressure sensors;mixing mechanisms, measuring the amount of tissue and/or fluid to bemoved along a particular pathway using volumetric mechanisms;maintaining temperatures of the various components using heat controldevices; washing and concentrating the cell, and integrating the processwith timing and software mechanisms. In one embodiment, software cancontrol the parameters of the process to allow production of a cellpopulation prepared to specific operator-defined parameters. Thus, theautomation device or devices improve the performance of the procedures,and provide automatic harvesting of adipose tissue and processing of theadipose tissue for administration to a patient.

One particular automation device is illustrated in FIG. 4. A tissueremoval container (not shown) is placed into a device 100 usingcolor-coded guide marks 112-118 to properly align and insert the tubinginto appropriate paths. Device 100 includes a plurality of valves 105and 110, and a plurality of pumps 104 and 109. Tubing is placed into aseries of valves 105, 110 and pumps 104, 109 which are controlled by anintegrated microprocessor system to coordinate fluid and tissue flow inaccordance with the user defined program. Program selection is mediatedthrough a user interface panel 106. A saline container is placed onto aholding structure 101 and attached to the tissue collection container. Avial or tube of collagenase or other tissue dissociation medium ormixture (not shown) is inserted into the tissue collection container atpoint 103. A waste bag (not shown) is inserted into a holding structure111, the cell separation chamber/cell collection container is placedinto a holding structure 107, and the tissue/cell mixing container isplaced into the holding structure 108. The tissue collection containeris placed into the agitation/incubation chamber 102.

Adipose tissue may be collected into the tissue collecting containerwhile the container is in position within the device or prior toplacement within the device. The device may contain an optionaltransparent insert 119 or other device allowing determination of thevolume of tissue within the tissue collecting container. Alternativelyvolume may be determined by measurement of the weight of materialcontained in the agitation/incubation chamber 102 (corresponding totissue collecting container 12). This volume may be displayed on theuser interface screen 106.

The microprocessor then opens the valves 105 on lines 114 and 115 andactivates the pumps 104 on line 114 for introduction of saline into thecollection chamber 102 and removal of waste material 115 to the wastebag 111. During this process the collection chamber is agitated byrocking, and is maintained at a programmed temperature by warmingdevices integrated into the chamber 102. In certain embodiments, tissueprocessing may use pre-warmed saline in which case the role of thewarming device of the agitation/incubation chamber is to maintaintemperature at the determined preprogrammed point rather than toincrease the temperature.

Once the tissue is washed some fraction from 0% to 100% of the intact,washed adipose tissue may be removed from the incubation chamber 102 byactivation of the pump 109 and valve 110 on line 116. Material withdrawnat this time is held in the mixing chamber 108. Dissociation medium 103is added to material remaining in the chamber 102 by opening the valve105 on line 113, closing other valves and activating pump 104 on line113. After addition of dissociation medium the chamber 102 is agitatedand maintained at temperature as described above. At the conclusion ofthe programmed incubation period agitation is halted to allow flotationof adipocytes. Additional saline may be added to facilitate thisprocess. Following flotation of adipocytes, the valves on lines 112 and115 are opened to allow removal of the target cell population from thechamber 102 into the cell washing chamber 107. Washed cells are removedthrough line 117 into the mixing chamber 108, supernatant and washingsolution are removed into the waste chamber 111 through line 118.Additional saline is passed into the system through line 114 to completethe washing process. Cells are mixed in the chamber 108 with any intacttissue removed through line 116 earlier in processing. Mixing may beachieved by any means known to those skilled in the art including butnot limited to agitation rocking/inversion of chamber, or by compressionpulsed or by moving rollers. Mixed material may then be removed throughthe port in the mixing chamber of the disposable set.

The device includes a microprocessor-controlled mechanism for automatingthe process according to pre-programmed parameters 106. This systemwould also include use of pressure sensors for detection of blockagesand similar safety and quality control mechanisms. In a preferredembodiment the software component of the system would include automatedcollection of “run data” including, for example, the lot numbers ofdisposable components, temperature and volume measurements, tissuevolume and cell number parameters, dose of enzyme applied, incubationtime, operator identity, date and time, patient identity, etc. In apreferred embodiment of the device a bar code reading system would beintegrated to permit data entry of these variables (for exampledisposable set lot number and expiration date, lot number and expirationdate of the Collagenase, patient/sample identifiers, etc.) into thedevice controller as part of documentation of processing. This wouldreduce the opportunity for data entry errors. This device could beeasily incorporated into the controller system using a USB or otherinterface port and system known to the art. In this way the device wouldprovide integrated control of the data entry and documentation of theprocess. A print-out report of these parameters would be part of theuser-defined parameters of a programmed operation of the device.Naturally this would require integration of a printer component(hardware and driver) or printer driver in software plus an interfaceoutput connector for a printer (e.g., a USB port) in the hardware of thedevice.

In a further embodiment, software incorporated into the controller wouldprompt users through the steps necessary for proper insertion of tubingand other elements into the device. Software would also initiateautomated testing to confirm correct insertion of tubing, absence ofblockages, etc.

The general approach to processing in this device would use the sameparameters as those described elsewhere in this disclosure for manualcell processing.

Many other conformations of the staged mechanisms used for cellprocessing will be apparent to one skilled in the art and the presentdescription is included as one example only. For example, mixing oftissue and saline during washing and disaggregation may occur byagitation as in the present example or by fluid recirculation. Cellwashing may be mediated by a continuous flow mechanism such as thespinning membrane approach, differential adherence, differentialcentrifugation (including, but not limited to differentialsedimentation, velocity, or gradient separation), or by a combination ofmeans. Similarly, additional components to allow further manipulation ofcells including addition of growth factors or other biological responsemodifiers (Lind, M., Growth factor stimulation of bone healing. Effectson osteoblasts, osteomies, and implants fixation. Acta Orthop ScandSuppl, 1998. 283: p. 2-37;Hanada, K., J. E. Dennis, and A. I. Caplan,Stimulatory effects of basic fibroblast growth factor and bonemorphogenetic protein-2 on osteogenic differentiation of rat bonemarrow-derived mesenchymal stem cells. J Bone Miner Res, 1997. 12(10):p. 1606-14; Lieberman, J. R., et al., Regional gene therapy with aBMP-2-producing murine stromal cell line induces heterotopic andorthotopic bone formation in rodents. J Orthop Res, 1998. 16(3): p.330-9), mixing of cells with other structural components (e.g., bonefragments (Jean, J. L., S. J. Wang, and M. K. Au, Treatment of a largesegmental bone defect with allograft and autogenous bone marrow graft. JFormos Med Assoc, 1997. 96(7): p. 553-7), collagen (Saadeh, P. B., etal., Repair of a Critical Size Defect in the Rat Mandible UsingAllogenic Type I Collagen. J Craniofac Surg, 2001. 12(6): p. 573-579)and/or synthetic components intended for implant with the cells into therecipient (Petite, H., et al., Tissue-engineered bone regeneration. NatBiotechnol, 2000. 18(9): p. 959-63.taf/dynapage.taf?file=/ncb/biotech/v18/n9/full/nbt0900_(—)959.htmltaf/dynapage.taf?file=/ncb/biotech/v18/n9/abs/nbt0900_(—)959.html;Gao,J., et al., Tissue-Engineered Fabrication of an Osteochondral CompositeGraft Using Rat Bone Marrow-Derived Mesenchymal Stem Cells. Tissue Eng,2001. 7(4): p. 363-71; Ohgushi, H. and A. I. Caplan, Stem celltechnology and bioceramics: from cell to gene engineering. J BiomedMater Res, 1999. 48(6): p. 913-27; Caplan, A. I. and V. M. Goldberg,Principles of tissue engineered regeneration of skeletal tissues. ClinOrthop, 1999(367 Suppl): p. S12-6). Post-processing manipulation mayalso include cell culture (Caplan, A. I. and S. P. Bruder, Mesenchyrnalstem cells: building blocks for molecular medicine in the 21st century.Trends Mol Med, 2001. 7(6): p. 259-64; Petite, supra; Zuk, P. A., etal., Multilineage cells from human adipose tissue: implications forcell-based therapies. Tissue Eng, 2001. 7(2): p. 211-28), gene transfer(Luskey, B. D., et al., Gene transfer into murine hematopoietic stemcells and bone marrow stromal cells. Ann N Y Acad Sci, 1990. 612(398):p. 398-406; Grompe, M., et al., Therapeutic trials in the murine modelof hereditary tyrosinaemia type I: a progress report. J Inherit MetabDis, 1998. 21(5): p. 518-31; Gazit, D., et al., Engineered pluripotentmesenchymal cells integrate and differentiate in regenerating bone: anovel cell-mediated gene therapy. J Gene Med, 1999. 1(2): p. 121-33;Mosca, J. D., et al., Mesenchymal stem cells as vehicles for genedelivery. Clin Orthop, 2000(379 Suppl): p. S71-90), or further cellpurification (Greenberg, A. W. and D. A. Hammer, Cell separationmediated by differential rolling adhesion. Biotechnol Bioeng, 2001.73(2): p. 111-24; Mainwaring, G. and A. F. Rowley, Separation ofleucocytes in the dogfish (Scyliorhinus canicula) using density gradientcentrifugation and differential adhesion to glass coverslips. CellTissue Res, 1985. 241(2): p. 283-90; Schweitzer, C. M., et al.,Isolation and culture of human bone marrow endothelial cells. ExpHematol, 1995. 23(1): p. 41-8). Mechanisms for performance of suchfunctions may be integrated within the device shown in FIG. 4 or may beincorporated in separate devices.

In additional embodiments of the invention, tissue collected into aconventional adipose tissue trap could be transferred into a processingset designed for processing other tissues. For example, Baxter Inc.manufacture and sell a series of plastic bags and filters intended foruse in the setting of a bone marrow transplant harvest (“Bone MarrowCollection Kit with Flexible Pre-Filters and Inline Filters”, ProductCode, 4R2107, U.S. Pat. Nos. 4,346,703 and 5,724,988). This bag setcontains a large conical bag with an integrated 800 μm filter whichcould be used for washing the collected adipose tissue.v In this exampleadipose tissue fragments larger than 800 μm would be retained in thebag. These fragments could then be washed by repeated addition of saline(or other washing solution) followed by removal of waste materialthrough ports below the filter. Mixing could be achieved manually or byuse of a benchtop rocking device and warming could be applied by use ofa heating pad. Disaggregation could occur within the lumen of this bag.Following disaggregation cells would pass through the integrated 800 μmfilter (and optionally through one or more filters of smaller mesh sizeprovided with the kit) and collected into a collection bag (alsoprovided). This bag could then be placed into a centrifuge (e.g., aSorval RC-3C) where cells could be serially washed and concentrated.Cells could also be washed using existing cell washing devices (largelydeveloped for washing human blood products) such as those sold by BaxterInc (Cytomate or Baxter CS3000) or by Cobe Inc. (Cobe Spectra). Thedisposable elements may be integrated using the fittings provided by themanufacturer or they may be linked by use of a sterile connecting devicesuch as those manufactured by Terumo Inc. Similarly the mechanismsdescribed in this less integrated approach could be linked to a centralcontroller and assembled as components of a more integrated device. Aperistaltic pump or battery of pumps could be used to automate fluidflow with use of manual or automated clamping to open and close fluidpathways.

In a preferred embodiment of the invention, the tissue removal systemand processing set would be present in the vicinity of the patientreceiving the treatment, such as the operating room or out-patientprocedure room (effectively at the patient's bedside). This allowsrapid, efficient tissue harvest and processing, remove the opportunityfor specimen handling/labeling error and thereby allow for performanceof the entire process in the course of a single surgical procedure.

The following examples are provided to demonstrate particular situationsand settings in which this technology may be applied and are notintended to restrict the scope of the invention and the claims includedin this disclosure

EXAMPLE 1 Autologous Fat Transfer

Autologous fat transfer is a relatively common cosmetic and structuralprocedure involving the harvest of adipose tissue (fat) from onelocation and reimplantation in another location within the sameindividual (Coleman, S. R. (1995). “Long-term survival of fattransplants: controlled demonstrations.” Aesthetic Plast Surg 19(5):421-5; Coleman, S. R. (2001). “Structural fat grafts: the ideal filler?”Clin Plast Surg 28(1): 111-9; Coleman, W. P., 3rd (1991). “Autologousfat transplantation.” Plast Reconstr Surg 88(4): 736.). However, asindicated above, this procedure is frequently compromised byinconsistent engraftment such that the implanted material is fully orpartially resorbed or is replaced by scar tissue (Eremia, S. and N.Newman (2000). “Long-term follow-up after autologous fat grafting:analysis of results from 116 patients followed at least 12 months afterreceiving the last of a minimum of two treatments.” Dermatol Surg26(12): 1150-8). At least part of the loss of function can be attributedto necrosis of implanted fat tissue during the time it takes for newblood vessels to form and feed the implant. Thus tissue implanted intohighly vascular areas such as muscle beds shows better engraftment thanwhen implanted into less well perfused tissues (Guerrerosantos, J., A.Gonzalez-Mendoza, et al. (1996). “Long-term survival of free fat graftsin muscle: an experimental study in rats.” Aesthetic Plast Surg 20(5):403-8).

Processed lipoaspirate prepared as described in this disclosureaddresses this issue by supplementing the implant with additionalendothelial precursors and stem cells. Extracted adipose tissuefragments from inbred Wistar rats were mixed with processed lipoaspiratein accordance with the methods disclosed herein. This composition wasthen implanted subcutaneously into the thigh and under the scalp ofrecipient rats. As controls an equal number of animals received adiposetissue alone (no processed lipoaspirate) under the scalp while animalsreceiving an implant in the thigh had the contralateral thigh implantedwith adipose tissue alone. Grafts were harvested one monthpost-implantation.

The results (FIG. 5) show a trend of increasing graft weight of thighimplants with increasing dose of processed lipoaspirate. Histologicexamination of the implants showed improved vascularity of graftssupplemented with processed lipoaspirate. A similar correlation wasobserved with scalp implants albeit with lower overall retention due tothe low vascularity of the dorsal skull in these rats.

In a clinical application of this technology, processed lipoaspiratederived according to this disclosure is prepared and mixed with intact(non-disaggregated) adipose tissue fragments, as disclosed above. Thecomposition comprising the mixture of adipose tissue and the stem cellsmay be implanted into the recipient to provide an autologous soft tissuefiller for correction of contour defects (wrinkles, “divots,” pockmarks,and larger deficits) (Coleman, S. R. (2001). “Structural fat grafts: theideal filler?” Clin Plast Surg 28(1): 111-9) or for providing support todamaged structures such as the urethra (Palma, P. C., C. L. Riccetto, etal. (1997). “Repeated lipoinjections for stress urinary incontinence.” JEndourol 11(1): 67-70; Lee, P. E., R. C. Kung, et al. (2001).“Periurethral autologous fat injection as treatment for female stressurinary incontinence: a randomized double-blind controlled trial.” JUrol 165(1): 153-8).

EXAMPLE 2 Acute Liver Injury

Liver damage induced by intraperitoneal injection with allyl alcohol isa common model of periportal acute liver injury (Lee, J. H., Z. Ilic, etal. (1996). “Cell kinetics of repair after allyl alcohol-induced livernecrosis in mice.” Int J Exp Pathol 77(2): 63-72; Werlich, T., K. J.Stiller, et al. (1999). “Experimental studies on the stem cell conceptof liver regeneration. II.” Exp Toxicol Pathol 51(1): 93-8.; Yin, L., D.Lynch, et al. (1999). “Participation of different cell types in therestitutive response of the rat liver to periportal injury induced byallyl alcohol.” J Hepatol 31(3): 497-507). This model has been used todemonstrate the presence of a population of stem cells that is criticalto liver regeneration (Yavorkovsky, L., E. Lai, et al. (1995).“Participation of small intraportal stem cells in the restitutiveresponse of the liver to periportal necrosis induced by allyl alcohol.”Hepatology 21(6): 1702-12; Werlich, T., K. J. Stiller, et al. (1999).“Experimental studies on the stem cell concept of liver regeneration.II.” Exp Toxicol Pathol 51(1): 93-8). We modified this model in SwissWebster mice in which alcohol-induced injury was followed by injectionof processed lipoaspirate. Animals were sacrificed at one week and thelivers were removed, weighed, and prepared for histologic examination.The results (FIGS. 6 and 7) show substantially improved liver weight inthose animals receiving processed lipoaspirate. Histologic analysisshowed normal histology within the treated animals with no evidence ofleukocyte infiltrate or any other mechanism that might aberrantlyincrease liver weight.

In a clinical setting, a patient with liver damage would have adiposetissue extracted and processed according to this disclosure. Processedlipoaspirate could then be injected intravenously for systemic deliveryor targeted to the liver through the hepatic circulatory system.

EXAMPLE 3 Acute Heart Damage

Acute myocardial infarct (heart attack) results in ischemic injury tothe myocardium. Tissue damage can be minimized by reperfusion of thedamaged tissue and by regeneration of myocardial tissue (Murry, C. E.,R. W. Wiseman, et al. (1996). “Skeletal myoblast transplantation forrepair of myocardial necrosis.” J Clin Invest 98(11): 2512-23; Orlic,D., J. Kajstura, et al. (2001). “Bone marrow cells regenerate infarctedmyocardium.” Nature 410(6829): 701-5; Rajnoch, C., J. C. Chachques, etal. (2001). “Cellular therapy reverses myocardial dysfunction.” J ThoracCardiovasc Surg 121(5): 871-8; Strauer, B. E., M. Brehm, et al. (2002).“Repair of infarcted myocardium by autologous intracoronary mononuclearbone marrow cell transplantation in humans.” Circulation 106(15):1913-8). The bedside approach described in this disclosure would providea potentially superior source of regenerative cells in that cells couldbe provided in greater numbers and purity without the morbidityassociated with a marrow harvest.

EXAMPLE 4 Allogeneic Application for Inherited Disease

Horwitz et al have demonstrated that stem cells from bone marrow canprovide clinical benefit to patients with a non-hematopoietic disorder,specifically Osteogenesis imperfecta (Horwitz, E. M., D. J. Prockop, etal. (1999). “Transplantability and therapeutic effects of bonemarrow-derived mesenchymal cells in children with osteogenesisimperfecta.” Nat Med 5(3): 309-13; Horwitz, E. M., D. J. Prockop, et al.(2001). “Clinical responses to bone marrow transplantation in childrenwith severe osteogenesis imperfecta.” Blood 97(5): 1227-31). In thesestudies the authors attempted to compensate for the low number andfrequency of non-hematopoietic stem cells in marrow by growing cells inculture to expand and purify the MSC component. However, as mentionedabove, growing cells in culture is associated with substantial technicaland clinical concerns and the potential of adipose tissue, processed inaccordance with this disclosure, to provide a source a large number ofstem cells without requiring cell culture represents a potentialsubstantive improvement in patient care. In this model a suitablymatched donor unaffected by the genetic disease (normal genotype orasymptomatic carrier), preferably a sibling or other first degreerelative, would be the source of donor cells although use of unrelateddonors is within the scope of this invention. Cells would be extractedfrom the adipose tissue for infusion into a patient with an inheriteddisease resulting in compromised tissue function or regeneration.Examples include, but are not limited to Osteogenesis imperfecta,Duschenes Muscular Dystrophy (and other Muscular Dystrophies), inheritedretinal degenerative diseases, hereditary tyrosinemia, and numerousother inherited diseases.

A corollary of this is that application of gene therapy approaches inwhich autologous (self) processed lipoaspirate is modified by insertionof a gene into the stem cell compartment would obviate the need for anallogeneic (no-self) donor. Such an approach could also be used in thetreatment of infectious disease in which a novel gene is inserted intothe stem cells. For example an antisense or ribozyme construct could beused to generate cells capable of providing an anti-HIV effect. Thisapproach could also be used to generate stem cells capable of acting asdrug delivery vehicles.

REFERENCES

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Any feature or combination of features described herein are includedwithin the scope of the present invention provided that the featuresincluded in any such combination are not mutually inconsistent as willbe apparent from the context, this specification, and the knowledge ofone of ordinary skill in the art. For purposes of summarizing thepresent invention, certain aspects, advantages and novel features of thepresent invention have been described herein. Of course, it is to beunderstood that not necessarily all such aspects, advantages or featureswill be embodied in any particular embodiment of the present invention.Additional advantages and aspects of the present invention are apparentin the following detailed description and claims.

The above-described embodiments have been provided by way of example,and the present invention is not limited to these examples. Multiplevariations and modification to the disclosed embodiments will occur, tothe extent not mutually exclusive, to those skilled in the art uponconsideration of the foregoing description. Additionally, othercombinations, omissions, substitutions and modifications will beapparent to the skilled artisan in view of the disclosure herein.Accordingly, the present invention is not intended to be limited by thedisclosed embodiments, but is to be defined by reference to the appendedclaims.

A number of publications and patents have been cited hereinabove. Eachof the cited publications and patents are hereby incorporated byreference in their entireties.

1. A method of processing a cell population that comprisesadipose-derived stem cells, comprising: removing a first portion ofadipose tissue that comprises a cell population that comprisesadipose-derived stem cells from a patient; introducing the removed firstportion of adipose tissue that comprises said cell population thatcomprises adipose-derived stem cells into a self-containedadipose-derived stem cell processing unit configured to maintain aclosed pathway, wherein said self-contained adipose derived stem cellprocessing unit comprises: a tissue collection container that isconfigured to receive adipose tissue that is removed from a patient,wherein said tissue collection container is defined by a closed system;a first filter that is disposed within said tissue collection container,wherein said first filter is configured to retain a first component ofsaid unprocessed adipose tissue and pass a second component of saidunprocessed adipose tissue, such that said first filter separates saidfirst component from said second component, and wherein said firstcomponent comprises a cell population that comprises adipose-derivedstem cells and said second component comprises lipid, mature adipocytes,and saline; a cell collection container, which is configured to receivesaid first component comprising a cell population that comprisesadipose-derived stem cells from said tissue collection container,wherein said cell collection container is within said closed system; aconduit configured to allow passage of said first component comprising acell population comprising adipose-derived stem cells from said tissuecollection container to said cell collection container while maintaininga closed system; a cell concentrator disposed within said cellcollection container, which is configured to facilitate theconcentration of said first component comprising a cell population thatcomprises adipose-derived stem cells so as to obtain a concentratedpopulation of cells that comprises adipose-derived stem cells, whereinsaid cell concentrator comprises a centriftige or a spinning membranefilter; and an outlet configured to allow the aseptic removal of saidconcentrated population of cells that comprise adipose-derived stemcells; separating and concentrating said cell population that comprisesadipose-derived stem cells from said removed first portion of adiposetissue within said self-contained cell processing unit while maintainingsaid closed pathway to obtain a concentrated cell population thatcomprises adipose-derived stem cells; and mixing said concentrated cellpopulation that comprises adipose-derived stem cells with a secondportion of unprocessed adipose tissue from said patient.
 2. The methodof claim 1,wherein said first or second portion of adipose tissue thatis removed from said patient is lipoaspirate.
 3. The method of claim 1,wherein said first or second portion of adipose tissue that is removedfrom said patient is obtained by excisional lipectomy.
 4. The method ofclaim 1, further comprising a disaggregation step, wherein said cellpopulation that comprises adipose-derived stem cells contained in saidremoved first portion of adipose tissue is mechanically or enzymaticallydisaggregated from said mature adipocytes and connective tissue presentin said first portion of adipose tissue that was removed from saidpatient prior to separation.
 5. The method of claim 1, wherein said cellconcentrator comprises a spinning membrane filter.
 6. The method ofclaim 1, wherein said cell concentrator comprises an antibody.
 7. Themethod of claim 6, wherein said antibody is selected from the groupconsisting of AP2, CD3, CD 19, and CD11 b.
 8. The method of claim 1,wherein said cell concentrator comprises a centrifuge.
 9. The method ofclaim 4, wherein said disaggregation step comprises an enzymaticdigestion.
 10. The method of claim 9, wherein said enzymatic digestioncomprises a collagenase.
 11. The method of claim 9, wherein saidenzymatic digestion comprises a neutral protease.
 12. The method ofclaim 9, wherein said enzymatic digestion comprises trypsin.
 13. Themethod of claim 1, wherein said adipose-derived stem cells in saidconcentrated cell population that comprises adipose-derived stem cellsare at least 0.1% of the cellular component.
 14. The method of claim 1,wherein said adipose-derived stem cells in said concentrated cellpopulation that comprises adipose-derived stem cells are between about2% and about 12% of the cellular component.
 15. The method of claim 1,wherein said concentrated cell population that comprises adipose-derivedstem cells has a concentration of about 2 ×10₇ cells/100 ml of adiposetissue.
 16. The method of claim 1, wherein said concentrated cellpopulation that comprises adipose-derived stem cells comprisesendothelial precursor cells.
 17. The method of claim 1, furthercomprising contacting said concentrated cell population that comprisesadipose-derived stem cells with an additive.
 18. The method of claim 17,wherein said additive is a tissue or tissue fragment.
 19. The method ofclaim 18, wherein said tissue or tissue fragment is unprocessed adiposetissue.
 20. The method of claim 18, wherein said tissue or tissuefragment is a connective tissue or bone.
 21. The method of claim 17,wherein said additive is a biological or artificial scaffold.